Structural and Functional Investigation of a Multi drug Efflux Transporter QacA

Abstract

The emergence of multi-drug resistance in bacteria is a global health care challenge. One of the effective means of gaining antimicrobial resistance, among superbugs, is through expression of efflux pumps. Quaternary ammonium compound transporters, QacA/B, that are observed in Staphylococcus aureus strains are capable of transporting 30 chemically dissimilar monovalent and divalent cationic antibacterial compounds and dyes. The 14-TM (transmembrane) helix containing transporter QacA, belongs to the drug/H+ antiporter 2 (DHA2) family which is a part of major facilitator superfamily (MFS). MFS transporters are the largest superfamily of secondary active transporters. QacA utilizes the H+ gradient across the bacterial cell membrane for the uphill efflux of the cytotoxic compounds. QacA has two distinct TM domains, each of which consists of 6-TM helices that retain a pseudo 2-fold symmetry amongst them. During the transport process, the domains move in rocker-switch mechanism to allow alternating-access to either side of the membrane in order to transport the substrates. The expression of this efflux pump is regulated by a trans-acting regulatory DNA binding protein QacR. Under normal condition, QacR blocks the transcription of qacA gene by binding to the operator DNA but under antibacterial stress, the substrates of QacA binds to QacR, causing dissociation of QacR from the operator DNA and QacA gets expressed. In this thesis, structural and functional investigation of QacA was carried out and some fundamental questions about this multi-drug efflux transporter are addressed. In the first part, wild-type QacA was purified and the transport activity of the transporter both in native membrane and in isolation was analyzed using substrate-induced H+-release assay and a reconstitution-based assay. The binding studies with the cytotoxic substrates (TPP, Pm, Dq) displayed sub-millimolar binding affinity with the purified transporter and substrate/H+ competition assay suggested the presence of substrate-protonation site...